The vitamin K-dependent (VKD) carboxylase is critical to hemostasis because it converts Glus to carboxylated Glus in VKD proteins to allow their binding to cell surfaces where hemostasis occurs. VKD proteins are carboxylated in the endoplasmic reticulum during their secretion, and a single carboxylase modifies all VKD proteins, many of which are coexpressed in tissue. In the previous grant period, we developed an approach to directly analyze intracellular carboxylation in mammalian cells, which showedthat the secretory process impacts carboxylation, that intracellular processing is not identical for all VKD proteins and that carboxylation is regulated by the availability of the reduced vitamin K cofactor required for Glu carboxylation. The studies also showed that the rate-limiting step in VKD turnover is different in cells than in an in vitro reaction, which may be due to post-translational modifications in the carboxylase. Other studies revealed that Leptospira, the bacterial pathogen that causes leptospirosis, contains an ortholog of the VKD carboxylase, which appears to have been acquired by horizontal gene transfer and to have been adapted for a role other than carboxylation. Our studies also implicated novel functional carboxylase residues, including the catalytic base that initiates carboxylation and residues whose substitution cause combined VKD coagulation factor deficiency. Our long-term goal is to understand the mechanism of carboxylation, including how it interfaces with the secretory machinery and how multiple VKD proteins are modified by one carboxylase to become fully-carboxylated and active. We propose: 1. To determine how the active site facilitates carboxylation. We will identify the catalytic base that initiates carboxylation and will determine how substitutions in carboxylase residues cause combined VKD factor deficiency. 2. To determine if turnovers of VKD proteins differ and are impacted by a second site of VKD protein-carboxylase interaction. We will determine whether the VKD proteins factor X and prothrombin are carboxylated with equal efficiencies, and whether having two sites of VKD protein-carboxylase interaction impacts efficiency. 3. To test our hypothesis that post-translational carboxylase modifications are important to VKD protein turnover. Sites of post-translational modification in the carboxylasewill be identified and mutated to determine if they impact VKD protein turnover. These studies will make important contributions to understanding carboxylation, which will be significant for developing superior anticoagulants and for producing VKD proteins for therapies in hemophilia and sepsis. Lav abstract. Vitamin K in the diet is used to activate a set of factors critical to blood clotting, and therefore it is important to understand how they become activated. The studies will impact the development of anticoagulants and the production of therapeutic proteins for treating hemophilia and septic shock.